RU1792615C - Method of beta-exotoxin producer cultivation - Google Patents

Abstract

Usage: biotechnology, the production of insecticides. SUMMARY OF THE INVENTION: producer of beta-exotoxin, a bacterial strain of You. thurlnglensls 98-1 s are cultured on a medium containing carbon and nitrogen sources to the stage of physiological activity of producer growth, in particular for 16 hours, then barium chloride in an amount of 0.6-0.7 mg / l or calcium chloride in an amount of 1.0-1.1 mg per 1 liter of culture medium and continue cultivation until the culture enters the stationary phase of growth. 1 tab.

Description

The invention relates to the field of biotechnology, in particular to methods for the production of biological insecticidal preparations and can be used for the production of SER or animal welfare.

A number of strains of the entomopathogenic microorganism Bacillus b1b1d1e1 $ 15 (hereinafter BT) during their cultivation release water-soluble exotoxin into the culture fluid. The yield of exotoxin depends on the composition of the nutrient media and can be increased by appropriate selection.

To precipitate exotoxin from BT culture fluid after complete maturation of the spores, a concentrated exotoxin method is proposed. For this, barium or calcium exotoxin binding salts are added to the filtrate (or to the centrate) of the culture fluid (CL).

The aim of the present invention is to increase the yield of beta-exotoxin

relatively economical means and reducing the time of fermentation.

Closest to the proposed method for cultivating a beta-exotoxin producer is a method for producing a bio-toxibacillin, which comprises cultivating an exotoxin producer strain in a medium containing a carbon source, nitrogen in the presence of a salt forming an insoluble complex with beta-exotoxin. As the latter, calcium acetate is used. The maximum achieved exotoxin yield when implementing this method is 1232-1480 mg / l with a calcium acetic acid flow rate of up to 65 g / l (6.5 wt.%).

Our goal is to increase the yield of beta-exotoxin with a decrease in the consumption of salts that bind beta-exotoxin to a water-insoluble complex, as well as to reduce the duration of the fermentation cycle by precipitating exotics

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sine released during BT cultivation by adding 0.6-0.7 mg of BaCte per 1 liter of QoL or 1.0-1.1 mg of CaClz per 1 liter of QoL in the BT culture fluid at the stage of maximum physiological activity. Moreover, if a preparation containing only exotoxin is needed, the fermentation process is stopped with the release of the culture to a stationary state of growth.

These additives form stable metal-exotoxin complexes and thereby remove the latter from the reaction. In this case, on the one hand, the inhibitory effect of exotoxin on the growth of BT microorganisms is reduced, and on the other hand, bound exotoxin cannot be consumed for secondary metabolism of bacteria.

Barium or calcium salts should be added for 10-16 hours of cultivation, which corresponds to the zone of maximum growth rate of microorganisms, since at this time the rate of exotoxin accumulation in QOL is also maximum.

The use of barium salts is most preferred since the degree of binding of this metal to exotoxin is greatest.

The exotoxin accumulation process ends at the stage when the culture reaches the stationary state of the growth curve; therefore, further cultivation is not required when only the exotoxin-containing preparation is obtained.

Upon preparation of a mixed exotoxin and exotoxin-containing preparation, cultivation is carried out based on the degree and rate of accumulation of endotoxin.

The following are examples of specific designs.

EXAMPLE 1-14 The cultivation of strain BT 98-1c was carried out in the AN-KUM-2 fermenter on a composition medium: protein-vitamin concentrate (BVC) 3.6% cornmeal 2.8% soya flour 0.5% chalk 0.3% The initial pH of the medium was 7.2 medium volume 1.0 l stirring speed 400 rpm air consumption for aeration 1 l / s temperature 30 ° C. In all variants of the experimental conditions, cultivation began with the ANKUM-2 apparatus switched to the mode. Rinse with a sterile saline solution with a volume of 30 ml from a braid of culture VT-98-1c, heated in a thermal bath at a temperature of 80 ° C for 5 min, was used as an inoculum. The cultivation was carried out with control of temperature, pH of the medium, p Oa of the cultivation time.

The physiological state of the cells was monitored by microscopy of the samples taken. Additives of an aqueous solution of barium chloride or an aqueous solution of barium chloride or an aqueous solution of calcium chloride, as well as the duration of the experiment, were carried out according to the experimental conditions scheme 1-14.

At the end of each fermentation, QoL analysis for exo

toxin in sediment, in solution and total

exotoxin content. The exotoxin content was determined using thin layer chromatography and spectrophotometry according to the procedure (3). The measurement error of the exotoxin content was ± 0.05 g / l. The experiments were carried out according to the following scheme:

1. Cultivation of BT 98 under the conditions described above, no additives were added during the cultivation of 5 to 20 hours (control).

2. Cultivation of BT 98-1 s as in (1), but 1.8 ml of a 30% aqueous solution of barium chloride (0.5 mg) per 1 l of QOL were added after 16 hours of cultivation.

0 s The same as (1), but 2.0 ml of a 30% aqueous solution of barium chloride (0.6 mg) per 1 L of QOL were added after 16 hours of cultivation.

4. Same as (1), but 2.3 ml of 30 5% aqueous solution of barium chloride (0.7 mg) per 1 L of QOL were added after 16 hours of cultivation.

5. The same as (1), but 1.5 ml of a 60% aqueous solution of calcium chloride (0.20 centigrade) (0.9 mg) per 1 liter of QoL were added after 16 hours of cultivation.

6. The same as (1), but 1.7 ml of a 60% aqueous solution of calcium chloride (1.0 mg) per 1 liter of QoL were added after 16 hours of cultivation. 5 days.

7. Same as (1), but 1.9 ml of a 60% aqueous solution of calcium chloride (1.1 mg) per 1 liter of QOL were added after 16 hours of cultivation.

08. Cultivation of BT 98-1c, as (1), but the cultivation time is 48 hours (control).

9. 9, 10, 11, 12, 13, 14 is the same as 2, 3, 4, 5, 6, 7, respectively, but the cultivation time is 48 hours.

5 The experimental results are presented in the table.

It follows from the table that the content of exotoxin in QOL after 20 hours of cultivation exceeds the content of exotoxin in QOL after 48 hours of cultivation by 45%.

The addition of barium chloride solution to the QOL for 16 hours (after a 20-hour cultivation) increases the exotoxin content by 65%, and the calcium chloride solution by 36%. The addition of barium or calcium chlorides for 16 hours (with 48-hour cultivation) also contributes to an increase in the exotoxin yield in comparison with the control, respectively, from 72% (BaCte) to 46% (CaCte), however, the exotoxin yield is quantitatively the case is lower than the exotoxin content recorded during a 20-hour cultivation with the same additives of barium or calcium chlorides.

Thus, to achieve the maximum concentration of exotoxin in the preparation, it is necessary to introduce the indicated additives at 16 hours from the start of cultivation and complete the fermentation process at 20 hours.

With 48-hour fermentation, the number of viable spores does not depend on the introduction of metal salts, the content of exotoxin in the presence of the latter increases by about 1.5-2 times.

Our method of cultivating a producer of beta-exotoxin was also tested on a medium of a different composition, in particular, proposed by the authors of the applications.

The main components of the medium: sources of carbon, nitrogen, lysine were preserved according to the proposed formulation, and the salts forming the insoluble complex with beta-exotoxin were introduced not at the beginning of fermentation, as the inventors propose, but at the growth stage corresponding to the maximum physiological activity (10 -16 h), and instead of calcium acetic acid, either calcium or barium salts were used in the concentrations proposed by us in this application (Examples 15 and 16).

PRI me R 15. The strain VT98-1c was grown on a medium of 1 liter containing protein-vitamin concentrate (BVK) - 3%, fodder yeast hydrolyzate with soluble protein content of 50 mg / l, rapeseed meal meal hydrolyzate with soluble content protein 8 mg / l, crystalline lysine (0.002 wt.%) at 30 ° C, aeration 1 l / min, stirring speed 400 rpm. For 10-16 hours of cultivation, which corresponded to the maximum physiological activity of the producer, 2 ml of a 30% aqueous solution of barium chloride were added. At 20 hours, the cultivation process was stopped.

The content of dissolved exotoxin in QL was: in the sediment - 0.92 g / l, in solution - 0.73 g / l, in total - 1.65 g / l.

Example 16. Strain BT 98-1c was cultured on an medium of the same composition as in Example 15. After 16 hours from the start of cultivation, 1.7 ml of a 60% aqueous solution of calcium chloride were added. For 20 hours of cultivation, the exotoxin content was: in the sediment 0.9 g / l, in the solution 0.6 g / l, the total amount 1.5 g / l.

Using the proposed method for producing exotoxin during BT cultivation provides the following advantages: if all the possibilities for increasing the exotoxin yield by optimizing the composition of the media in this strain are exhausted, the yield can be increased up to 70% by adding BaCla (or CaCte) solution for approximately 16 hours of growth and terminating the process cultivation for 20 hours

Thus, the use of the proposed method for producing exotoxin provides the following advantages: an increase in exotoxin yield by 70%, a decrease in fermentation time by 45%, and a significant reduction in salt consumption, binding beta-exotoxin to a water-insoluble complex.

The economic effect of the implementation of the invention will be provided by increasing the yield of exotoxin and reducing technological costs during fermentation, and will be at least 700 thousand rubles across the USSR. per year with a profitability level of 33%.

Claims (1)

SUMMARY OF THE INVENTION A method of cultivating a beta-exotoxin producer on a nutrient medium containing carbon and nitrogen sources in the presence of a salt forming an insoluble complex with beta-exotoxin, characterized in that, in order to increase the yield of the target product while reducing salt consumption and cultivation time, barium chloride in an amount of 0.6-0.7 mg / l or calcium chloride in an amount of 1.0-1.1 mg per 1 liter of culture fluid is used as the salt; moreover, the salt is introduced during cultivation at the stage of maximum physiological activity growth of the producer, and the cultivation process is carried out until the culture enters the stationary phase. CONTENTS OF EXOTOXIN IN QOLES DEPENDING ON THE ADDITION OF METAL SALT